Experiments in this application will examine the molecular mechanisms responsible for the modulation of cellular metabolism and resistance to oxidants by endogenous nitric oxide (NO). Published data indicated that NO either directly mainly by reversible S-nitrosylation of critical cysteine residues or by elevating cGMP levels modulates the adaptive responses that render cells resistant to oxidative stress and apoptosis. However, the majority of the cellular models rely upon the deliver of NO by NO donors or by the induction of the inducible nitric oxide synthase (NOS). To study the contribution of NO generated by the low output endothelial NOS in the cellular protection against oxidants, we utilized ECV3O4 cells transfected with endothelial NOS. The transfected cells generated sufficient NO to induce elevation of cGMP in smooth muscle cells in an L-NAME inhabitable manner. Using this well-defined model preliminary data revealed that NO regulates the steady state of ATP, the flux of glucose by the glycolytic and pentose phosphate pathways and respiration. Moreover, this dynamic regulation of metabolism and mitochondrial bioenergetics was associated with an increased resistance to H2O2 exposure. Exposure to H2O2 at 50-100 pM induced a delayed cell death (18 hours after exposure) to nearly 50 percent of ECV3O4 but less than 20 percent in the ECV3O4-eNOS cells. Inhibition of NO production ameliorated the protective effect and restored the steady state levels of ATP and glucose fluxes. Preliminary data using human pulmonary artery endothelial cells confirmed the NO-dependent protection against H202 induced delayed cell death. These preliminary data together with scarce published data on the ability of NO to regulate metabolism suggest a previous unrecognized function of NO that may causally relate to adaptation against oxidative stress. We propose that the generation of low levels of NO by eNOS is sufficient to dynamically regulate cellular glucose metabolism and respiration providing a primary and previously unrecognized molecular mechanism for the NO-induced protection against oxidative stress. To examine these hypotheses we propose the following specific aims: (1) define the molecular mechanism(s) of nitric oxide-mediated regulation of cellular metabolism; (2) investigate the causal association between nitric oxide-dependent alterations in metabolism with the adaptation to oxidative stress; and (3) examine if endogenous nitric oxide regulation of mitochondrial respiration and mitochondrial function is responsible for the protection against oxidative stresses. Experiments in the first aim are focused on the allosteric, covalent and other regulatory functions of NO in critical enzymes that catalyze essential and irreversible steps in the glycolytic pathway and TCA cycle. The second aim will utilize biochemical, pharmacological and molecular approaches to provide evidence for the potential causal relationship between NO-mediated regulation of metabolism and resistance to oxidative stress. The third aim examines the importance of NO-regulated mitochondrial respiration and function in protecting cells from oxidant exposures and typical inducers of apoptosis. Overall the proposed experiments will evaluate in a systematic manner the critical role of endogenously generated NO as a mediator of cellular metabolism and respiration that enables cells to resist oxidative stress.